WO2012098864A1 - 抗アポトーシス又は抗ネクローシス誘導方法 - Google Patents

抗アポトーシス又は抗ネクローシス誘導方法 Download PDF

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Publication number
WO2012098864A1
WO2012098864A1 PCT/JP2012/000252 JP2012000252W WO2012098864A1 WO 2012098864 A1 WO2012098864 A1 WO 2012098864A1 JP 2012000252 W JP2012000252 W JP 2012000252W WO 2012098864 A1 WO2012098864 A1 WO 2012098864A1
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WO
WIPO (PCT)
Prior art keywords
cells
effect
necrosis
voltage
apoptosis
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Application number
PCT/JP2012/000252
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English (en)
French (fr)
Japanese (ja)
Inventor
正太 小玉
友行 五代
Original Assignee
学校法人福岡大学
株式会社マヤテック
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 学校法人福岡大学, 株式会社マヤテック filed Critical 学校法人福岡大学
Priority to US13/993,832 priority Critical patent/US9206415B2/en
Priority to CN201280004292.5A priority patent/CN103261406B/zh
Priority to JP2012553616A priority patent/JP5819861B2/ja
Priority to KR1020137008840A priority patent/KR101549573B1/ko
Priority to EP12736365.3A priority patent/EP2666852B1/en
Publication of WO2012098864A1 publication Critical patent/WO2012098864A1/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • the present invention relates to a method for inducing an anti-apoptotic effect and / or an anti-necrosis effect in living cells.
  • Apoptosis is a type of death of cells that make up the body of a multicellular organism, and cell suicide that is actively triggered to keep an individual in a better state, that is, programmed cell death.
  • cell death caused by deterioration of the environment inside and outside the cell due to poor blood circulation, trauma, etc. is known as necrosis.
  • various drugs are administered to living cells to induce anti-apoptotic effect or anti-necrosis effect. It has never been known at all to induce an anti-apoptotic effect or an anti-necrosis effect. JP 2004-305046 A International Publication No. 2008/096631
  • the present invention aims to provide a method for inducing an anti-apoptotic effect and / or an anti-necrosis effect in living cells easily and with good control without administering a drug. is there.
  • the present invention relates to a method for inducing anti-apoptosis and / or anti-necrosis in living cells, wherein an AC voltage is applied to the living cells so that a current of 25 ⁇ A or more and 75 ⁇ A or less flows.
  • cultured cells can be used as the living cells.
  • the voltage value of the AC voltage is preferably 10 V or more and 5 kV or less.
  • the present invention can be implemented by applying the AC voltage to a mounting member on which a container holding the living cells is mounted.
  • an anti-apoptotic effect and / or an anti-necrosis effect can be induced in a living cell easily and with good control without administering a drug.
  • the schematic diagram which shows the structure of the alternating voltage application apparatus which can be used by one Embodiment of this invention.
  • FIG. 1 is a schematic diagram showing the structure of an AC voltage application device that can be used in an embodiment of the present invention.
  • the AC voltage application device 1 has a treatment tank 2.
  • This treatment tank may be a sealable culture device used in normal cell culture.
  • the culture apparatus enables cell culture by keeping the temperature and humidity therein constant.
  • the processing tank 2 has a box shape, and the wall portion is made of a conductive material. A part of the wall of the treatment tank 2 is provided with a door 2a for taking in and out the container 6 holding live cells.
  • a cradle 5 is disposed inside the treatment tank 2.
  • the cradle 5 is, for example, a support member 4 made of an electrically insulating material and erected on the bottom surface of the treatment tank 2, and a plate-like placement made of a conductive material disposed at the tip of the support member 4.
  • the container 6 is placed on the placement member 3.
  • the container 6 is usually a container having a depression on the upper surface, and live cells are accommodated and held in the depression.
  • a glass or resin dish for holding live cells is generally used.
  • the AC voltage application device 1 has a transformer 7, and one terminal 9 of the pair of secondary output lines of the transformer 7 is insulated and is not connected anywhere.
  • the load impedance is smaller than when the output terminal is open. Therefore, the output voltage of the transformer 7 can be lowered.
  • the other of the pair of secondary terminals of the transformer 7 is connected to the mounting member 3 of the table 5 of the processing tank 2 through the safety resistor R ⁇ b> 0 by the wiring 11.
  • the wiring 11 is electrically insulated from the wall of the processing tank 2 as appropriate.
  • the primary voltage V1 can be adjusted with the voltage operation part which is not shown in figure, and, thereby, the load voltage VL applied to the container 6 can be adjusted.
  • the door 2a of the treatment tank 2 is opened and closed, and the container 6 holding live cells is placed on the mounting member 3 of the table 5.
  • the primary voltage V ⁇ b> 1 is applied between the pair of primary terminals of the transformer 7.
  • This primary voltage V1 is a commercial frequency sinusoidal AC voltage here.
  • the secondary voltage V2 is induced between the secondary side terminals of the transformer 7, and the load voltage VL obtained by subtracting the voltage drop due to the safety resistance R0 from the secondary voltage V2 is applied to the container 6. Since the container 6 and the insulated terminal 9 are insulated from each other by the support member 4 and the air between the secondary terminals, the secondary circuit of the transformer 7 and the container 6 are connected to the container 6.
  • This load current is very weak. This is because the load impedance between the container 6 and the air between the secondary terminals is very large. The weak load current suppresses the occurrence of apoptosis and / or necrosis in the live cells held in the container 6.
  • the present invention is characterized by using a weak current.
  • an anti-apoptotic effect and / or an anti-necrosis effect in living cells can be induced in a very narrow range where the current value of the load current is 25 ⁇ A or more and 75 ⁇ A or less. Most preferably, it is around 50 ⁇ A (40 ⁇ A to 60 ⁇ A).
  • the present invention uses an alternating voltage, and the direct voltage cannot induce an anti-apoptotic effect and / or an anti-necrosis effect.
  • the energization time for inducing the anti-apoptotic effect and / or anti-necrosis effect is not particularly limited. Although a corresponding effect can be obtained even in a short time, it is preferably 12 hours or longer, more preferably 24 hours or longer. If the energization time is long, there is a risk of inducing apoptosis or necrosis on the contrary. For example, it is preferably within 48 hours.
  • the living cells that can be used in the present invention are not particularly limited, but living cells collected from multicellular organisms such as mammals such as humans, mice, and rats, and birds can be used. Moreover, the living cell currently culture
  • the culture conditions are not limited.
  • An appropriate temperature, humidity, atmosphere, culture solution, and additive may be selected according to the type of living cells.
  • an anti-apoptotic effect and / or an anti-necrosis effect can be suitably induced by passing a weak current while culturing cultured cells under normal conditions.
  • Culturing was performed in a tissue / cell culture vessel manufactured by Thermo.
  • An electrically insulating support member was disposed in the incubator, and a conductive placement member was further disposed thereon.
  • the above-mentioned dish was allowed to stand on this mounting member, and a depak made by Santec Engineering Co., Ltd., which is the above-described energization device, was connected to the above-described mounting member.
  • the culture conditions were set to a temperature of 37 ° C., a carbon dioxide of 5%, and a humidity of 100% throughout, and the cells were cultured in this environment regardless of whether or not energized. Further, careful attention was paid to the connection of the energization device in the incubator, and it was frequently confirmed that there was no interference in the energization region using the detector VoltAlert (Fluke, Japan, www.fluke.com).
  • the current setting of the energization device is set to 4 groups of 0 ⁇ A (no energization), 25 ⁇ A, 50 ⁇ A, and 75 ⁇ A, and the voltage is set in the range of 10V to 10000V, and each test cell is energized accurately for 24 hours.
  • Cultured The energized cultured cells were quickly washed three times with a washing solution at 4 ° C., and the cells were physically detached from the culture dish using a cell scraper (Fisher Scientific, Pittsburgh, PA) and collected in a microcentrifuge tube. After centrifugation at 4 ° C., the supernatant was removed, and mRNA was extracted from each group of cells and weighed.
  • the gene expression was quantified as to how many times the other groups showed higher expression than the control group of 0 ⁇ A (no energization), and are shown in Tables 1 and 2.
  • the gene with increase or attenuation is It is as follows.
  • Bcl10 increases to show anti-apoptotic effect
  • Bcl2 / 10 decreasing shows an apoptotic effect
  • Naip1 decreasing shows an apoptotic effect
  • Casp14 increases to show apoptotic effect
  • Hells increase shows anti-apoptotic effect
  • IL10 increases to show anti-apoptotic effect
  • Lxh4 increases to show anti-apoptotic effect
  • Nme5 increases to show anti-apoptotic effect
  • Trp63 increases to show apoptotic effect
  • tissue staining method chamber assay method 4-1) AnnexinV x PI staining AnnexinV (green) for detecting apoptotic cell membrane by engrafting NIH3T3 cells in the chamber by the culture method described above ) And Propidium Iodido (PI) (red) for detecting necrotic cells.
  • the set current of the energization device was set to 4 groups of 0 ⁇ A (no energization), 25 ⁇ A, 50 ⁇ A, and 75 ⁇ A.
  • the proportion of PI positive cells after energization decreased in the 25 ⁇ A, 50 ⁇ A, and 75 ⁇ A groups compared to the control group of 0 ⁇ A (no energization). That is, it was shown that the anti-necrosis effect was induced by energization.
  • LC3xDAPI staining NIH3T3 cells were engrafted in the chamber by the culture method described above, and co-stained with LC3 protein (red) that appeared when necrosis protein was induced and DAPI (blue) that detected the cell nucleus.
  • the proportion of LC3-positive cells decreased after energization in the 25 ⁇ A, 50 ⁇ A, and 75 ⁇ A groups with respect to the control group of 0 ⁇ A (no energization).
  • the ratio of LC3-positive cells was 50%, whereas it was observed that the ratio was significantly reduced to 28% in the 25 ⁇ A group and 16% in the 50 ⁇ A group. Even in this system, it was shown that the anti-necrosis effect was induced by energization.
  • NIH3T3 cells were engrafted in the chamber by the culture method described above and co-stained with Annexin V (green) for detecting apoptotic cell membranes and Propidium Iodido (PI) (red) for detecting necrotic cells.
  • the set current of the energization device was set to 4 groups of 0 ⁇ A (no energization), 25 ⁇ A, 50 ⁇ A, and 75 ⁇ A.
  • the set current of the energization device was set to 15 ⁇ A or 100 ⁇ A, and the anti-apoptotic effect and the anti-necrosis effect were confirmed using the flow cytometry method in the same manner.
  • the proportion of necrotic cells was approximately the same in the 15 ⁇ A or 100 ⁇ A group compared to the control 0 ⁇ A group.
  • a slight decrease in the proportion of apoptotic cells was observed, but the degree of decrease was small when compared in the 25 ⁇ A to 75 ⁇ A group. That is, in the group of 15 ⁇ A or 100 ⁇ A, no significant induction of anti-apoptotic effect and anti-necrosis effect was confirmed.
  • the present invention it is possible to retain living cells while suppressing the occurrence of apoptosis or necrosis in the living cells. In particular, it becomes possible to suppress the occurrence of apoptosis or necrosis in cultured cells.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2012/000252 2011-01-19 2012-01-18 抗アポトーシス又は抗ネクローシス誘導方法 WO2012098864A1 (ja)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US13/993,832 US9206415B2 (en) 2011-01-19 2012-01-18 Anti-apoptosis or anti-necrosis induction method
CN201280004292.5A CN103261406B (zh) 2011-01-19 2012-01-18 抗细胞凋亡或抗细胞坏死的诱导方法
JP2012553616A JP5819861B2 (ja) 2011-01-19 2012-01-18 アポトーシス又はネクローシスの発生抑制方法
KR1020137008840A KR101549573B1 (ko) 2011-01-19 2012-01-18 아폽토시스 또는 네크로시스 저해 방법
EP12736365.3A EP2666852B1 (en) 2011-01-19 2012-01-18 Anti-apoptosis or anti-necrosis induction method

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JP2011008828 2011-01-19
JP2011-008828 2011-01-19

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WO2012098864A1 true WO2012098864A1 (ja) 2012-07-26

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US (1) US9206415B2 (ko)
EP (1) EP2666852B1 (ko)
JP (1) JP5819861B2 (ko)
KR (1) KR101549573B1 (ko)
CN (1) CN103261406B (ko)
TW (1) TWI535846B (ko)
WO (1) WO2012098864A1 (ko)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015040816A1 (ja) 2013-09-19 2015-03-26 株式会社サンテツ技研 冷凍品の解凍方法

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JP7257667B2 (ja) * 2019-02-19 2023-04-14 柴田科学株式会社 ４気筒式ダイヤフラムポンプ

Citations (4)

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JPH11511322A (ja) * 1995-08-25 1999-10-05 サイエンティフィック ジェネリクス リミテッド 細胞内物質の放出
JP2004305046A (ja) 2003-04-03 2004-11-04 Mayatec Inc 物の酸化抑制方法及び交流電圧印加装置
JP2006167476A (ja) * 2004-12-17 2006-06-29 Hakuju Inst For Health Science Co Ltd 電界による疾患治療方法
WO2008096631A1 (ja) 2007-02-06 2008-08-14 Mayatec Co., Ltd. 処理装置

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US5387231A (en) * 1992-07-21 1995-02-07 Sporer; Patsy Electrotherapy method
US5817138A (en) * 1996-11-27 1998-10-06 Suzuki; James Y. Multi-channel, interferential wave, micro current device and methods for treatment using micro current
EP1337625B1 (de) * 2000-11-29 2004-07-14 Albert Scheller Stammzellen, verfahren zu deren expansion in vitro sowie deren verwendung
JP3939555B2 (ja) * 2002-01-17 2007-07-04 株式会社インテクト 電場ユニット装置
CN2721133Y (zh) * 2003-12-31 2005-08-31 中国医学科学院放射医学研究所 脉冲电磁场肿瘤治疗装置
CN101878053B (zh) * 2007-10-30 2014-04-09 麦克内尔-Ppc股份有限公司 具有感觉提示的微电流装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11511322A (ja) * 1995-08-25 1999-10-05 サイエンティフィック ジェネリクス リミテッド 細胞内物質の放出
JP2004305046A (ja) 2003-04-03 2004-11-04 Mayatec Inc 物の酸化抑制方法及び交流電圧印加装置
JP2006167476A (ja) * 2004-12-17 2006-06-29 Hakuju Inst For Health Science Co Ltd 電界による疾患治療方法
WO2008096631A1 (ja) 2007-02-06 2008-08-14 Mayatec Co., Ltd. 処理装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2666852A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015040816A1 (ja) 2013-09-19 2015-03-26 株式会社サンテツ技研 冷凍品の解凍方法

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US9206415B2 (en) 2015-12-08
EP2666852B1 (en) 2016-04-13
KR101549573B1 (ko) 2015-09-03
US20130316429A1 (en) 2013-11-28
TWI535846B (zh) 2016-06-01
TW201300527A (zh) 2013-01-01
EP2666852A1 (en) 2013-11-27
JP5819861B2 (ja) 2015-11-24
EP2666852A4 (en) 2014-09-10
KR20130058746A (ko) 2013-06-04
JPWO2012098864A1 (ja) 2014-06-09
CN103261406A (zh) 2013-08-21
CN103261406B (zh) 2014-11-19

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